One miR to rule them all

Abstract

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects 30% of the global population. With rising numbers it presents a risk for global health and health care systems. The development of MASLD and the more severe stage, metabolic dysfunction-associated steatohepatitis (MASH), is driven not only by comorbidities like obesity and type 2 diabetes, but also by hypothyroidism. Independent of the systemic thyroid hormone levels, dysregulation of hepatic thyroid hormone signaling plays an important role in MASLD pathogenesis, with the only approved medication for MASH treatment, resmetirom, being a thyroid hormone receptor beta (THRB) agonist. While the effect of thyroid hormone signaling dysregulation on the hepatic metabolism has been investigated thoroughly there is little information on whether epigenetic regulations contribute to this dysregulation in MASLD. Therefore, the aim of this thesis was the investigation of epigenetic regulations on hepatic thyroid hormone signaling in MASLD. Since the expression of THRB negatively correlates with the disease progression marker MASLD activity score (MAS) and since miRNAs have inhibiting effects on their target genes, miRNAs that potentially bind to THRB were identified. The expression of miRNAs and mRNAs potentially involved in the regulation of thyroid hormone action in the liver was measured in human and mouse liver tissue using qPCR. Verification of increased expression of potential THRB targeting miRNAs was performed in a cohort of obese individuals (BMI > 30 kg/m 2) which was divided into a non-MASH group (n = 41, MAS ≤ 3) and MASH group (n = 27, MAS ≥ 4), as well as in multiple mouse models mimicking different stages of MASLD. Binding of miRNA to target mRNAs was verified using luciferase reporter assays, as well as RNA-interacting protein immunoprecipitation-sequencing. Furthermore, cell culture models were metabolically stimulated to induce a MASLD-like state in the cells and investigate the effect on the expression of thyroid hormone metabolism genes. Additionally, DNA methylation was measured at THRB and miRNA genes, to further investigate involvement of additional epigenetic regulators. Unfortunately, no suitable THRB antibody was available to verify the results of mRNA measurements on the protein level. To address this, plasmids were designed to identify tags, that when added to THRB do not interfere with thyroid hormone signaling. miR-34a-5p was identified to be increased in MASLD and was predicted to bind not only THRB but also the thyroid hormone receptor alpha, the thyroid hormone activating enzyme DIO1 as well as the thyroid hormone transporters SLC10A1 and SCL16A2. The binding to THRB and DIO1 was verified and induced overexpression of miR-34a-5p reduced the levels of THRB regulated genes after stimulation with thyroid hormones. Cell culture experiments further showed induction of miR-34a-5p by stimulation with fatty acids. Interestingly, the results of the overexpression of miR-34a-5p on potential target genes could also be induced by stimulation with fructose and insulin without miR-34a- 5p involvement. Repression of thyroid hormone signaling through miR-34a-5p, reducing the expression of THRB as well as DIO1 which reduces the amount of available active thyroid hormone further, is exacerbated by increased DNA methylation in the THRB gene, which negatively correlates with THRB expression. Finally, treatment of high-fat diet fed mice with metformin reduced the expression level of miR-34a-5p. This reduced dysregulation of the miRNA could potentially recover THRB expression and signaling and might also be of use to patients unresponsive to resmetirom due to low THRB levels.